This application is the U.S. National Stage entry under 35 U.S.C. xc2xa7371 of PCT/FR97/01344, filed Jul. 8, 1997.
The present invention relates to novel synthetic polysaccharides possessing the anticoagulant and antithrombotic pharmacological activities of heparin.
Heparin belongs to the family of glycosaminoglycans (GAGs), which are heterogeneous natural sulphated polysaccharides.
Heparin preparations are mixtures of chains comprising a number of monosaccharide units ranging from 10 to 100 and more. In addition to this size heterogeneity there is a structural heterogeneity, as regards the nature of the constituent monosaccharides and also as regards the substituents which they bear (L. Rodxc3xa9n in: The Biochemistry of Glycoproteins and Glycosaminoglycans, Ed by Lennarz W. J., Plenum Press, New York and London, 267-371, 1980).
Each family of natural GAGs generally possesses a wide range of pharmacological activities. All are combined in the preparations which may be obtained from natural products. Thus, for example, the heparin and heparan sulphates possess an antithrombotic activity which is associated with the simultaneous action of several coagulation factors.
Heparin catalyses, in particular via antithrombin III (AT III), the inhibition of two enzymes involved in the blood coagulation cascade, namely factor Xa and factor IIa (or thrombin). Low molecular weight heparin (LMWH) preparations contain chains formed of 4 to 30 monosaccharides and have the property of acting more selectively on factor Xa than on thrombin.
Certain synthetic oligosaccharides, in particular those described in EP 84,999, have the property of selectively inhibiting, via antithrombin III, factor Xa without having any activity on thrombin.
It is known that the inhibition of factor Xa requires binding of the heparin to AT III via the antithrombin-binding region (ABR), and that the inhibition of factor IIa (thrombin) requires binding to AT (III), via ABR, as well as to thrombin via a less well-defined binding region (TBR).
The synthetic oligosaccharides corresponding to the ABR region of heparin are known and manifest an antithrombotic activity in venous thrombosis. These compounds are described in EP 529,715 and EP 621,282 and in Canadian patent 2,040,905.
The efficacy of these oligosaccharides in the prevention of arterial thrombosis is, nevertheless, hampered by their inability to inhibit thrombin.
A synthesis of glycoaminoglycans of heparin type which are capable of inhibiting thrombin via the AT (III) activator presents great difficulties and, indeed, this has never been achieved.
With the aim of rediscovering the activity of thrombin-inhibitor and factor Xa-inhibitor products, in EP-A-0,649,854 it has been proposed to connect two small oligosaccharides (an ABR and a TBR) by a species (xe2x80x9cspacerxe2x80x9d) which is not involved in the biological activity.
It has now been found that novel polysaccharide derivatives may be synthesized relatively simply and are biologically active. They are, in particular, anticoagulant and antithrombotic. Furthermore, on account of the production of these polysaccharides by synthesis, it is possible to selectively modify their structure, and in particular to remove unwanted sulphate substituents involved in the interaction with certain proteins. Thus, polysaccharides may be obtained which are powerful antithrombotic and anticoagulant agents and which may furthermore escape in vivo the action of proteins such as platelet factor 4 (PF4), which neutralize the effect of heparin in particular on thrombin.
Thus, it has been found, surprisingly, that sulphated and alkylated polysaccharides may be powerful antithrombotic and anticoagulant agents depending on the arrangement of the alkyl and sulphate groups borne by the carbohydrate skeleton.
More generally, it has been found that by preparing polysaccharide sequences, it is possible to modify with precision the GAG-type activities in order to obtain very active products which have the properties of heparin.
Thus, according to one of its aspects, the present invention relates to a novel synthetic polysaccharide comprising an antithrombin III-binding region consisting of a sequence of five monosaccharides bearing in total two carboxylic acid functions and at least four sulphate groups, this region being bound directly at its non-reducing end by a thrombin-binding region comprising a sequence of 10 to 25 monosaccharide units chosen from hexoses, pentoses or deoxy sugars in which all the hydroxyl groups are, independently, etherified with a (C1-C6)alkyl group or esterified in the form of sulphate groups, as well as its salts, in particular its pharmaceutically acceptable salts.
Preferably, the invention relates to a polysaccharide as defined above, characterized in that all its hydroxyl groups are etherified with a methyl or are esterified in the form of a sulpho group and its salts, in particular its pharmaceutically acceptable salts.
The products of the present invention are, in particular, polysaccharides represented by the following formula: 
in which
the wavy line denotes a bond either below or above the plane of the pyranose ring, 
xe2x80x83denotes a polysaccharide Po containing n identical or different monosaccharide units, which is linked via its anomeric carbon to Pe, 
xe2x80x83is a diagrammatic representation of a monosaccharide unit of pyranose structure chosen from hexoses, pentoses and the corresponding deoxy sugars, this unit being linked via its anomeric carbon to another monosaccharide unit, and the hydroxyl groups of this unit being substituted with identical or different groups xe2x80x94X, the groups X being chosen from (C1-C6)alkyl groups and sulpho groups,
n is an integer from 10 to 25,
Pe represents a pentasaccharide of structure: 
in which
R represents a (C1-C6)alkyl or a sulpho group,
R1a represents R1 or constitutes, with the oxygen atom to which it is attached and the carbon atom bearing the carboxylic function on the same ring, a group
Cxe2x80x94CH2xe2x80x94O,
R represents a (C1-C6)alkyl,
W represents an oxygen atom or a methylene group,
or one of their salts, in particular a salt which is pharmaceutically acceptable.
It will be noted in general in the present description that a wavy line denotes a bond either below or above the plane of the pyranose ring.
The monosaccharides contained in Po may be identical to or different from each other, and the interglycoside linkages may be of the xcex1 or xcex2 type.
These monosaccharides are advantageously chosen from the D or L hexoses allose, altrose, glucose, mannose, galose, idose, galactose and talose (in this case h=3) or from the D or L pentoses ribose, arabinose, xylose and lyxose (in this case h=2). Other monosaccharides such as, for example, deoxy sugars may also be used (h=1 and/or xe2x80x94CH2OXxe2x95x90CH3).
When, in the pentasaccharides Pe, the unit W represents an oxygen atom and R1a is as defined for R, these pentasaccharides constitute known compounds described in particular in patents EP 300,099, EP 529,715, EP 621,282 and EP 649,854 as well as in the literature. They are obtained from synthons which are also described in the literature by C. van Boeckel and M. Petitou, Angew. Chem. Int. Ed. Engl., 1993, 32, 1671-1690.
When, in the pentasaccharides Pe, R1a is other than R1 and/or W represents a carbon atom, these pentasaccharides are prepared using novel synthons which constitute a further aspect of the invention.
When, in the pentasaccharides Pe, the unit of L-iduronic acid type is replaced with a unit whose conformation is locked by a bridge, these pentasaccharides are prepared using novel synthons which constitute a further aspect of the invention.
Thus, according to another of its aspects, the present invention relates to novel intermediates which are useful for the preparation of compounds (I).
The polysaccharide part Po may consist of 10 to 25 alkylated and di- or trisulphated monosaccharide units.
The polysaccharide part Po may consist of 10 to 25 alkylated and mono- or disulphated monosaccharide units.
The polysaccharide part Po may consist of 10 to 25 uncharged and/or partially charged and/or fully charged alkylated monosaccharide units.
The charged or uncharged units may be dispersed along the entire length of the chain or they may, in contrast, be grouped in charged or uncharged saccharide regions.
The linkages may be 1,2; 1,3; 1,4; 1,5; 1,6; and of the xcex1 or xcex2 type.
In the present description, it has been chosen to represent the conformations 1C4 for L-iduronic acid and 4C1 for D-glucuronic acid, but it is well known that, in general, the conformation of the monosaccharide units in solution fluctuates. Thus, L-iduronic acid may be of 1C4 2S0 or 4C1 conformation.
Preferred compounds according to the invention are those of formula (I.A): 
in which 
denotes a particular family of polysaccharides Po, linked via their anomeric carbon to Pe as defined for 
is as defined for (I),
the OXs are as defined for (I) and, for the same polysaccharide, may be identical or different,
the monosaccharides contained in [ ]m form a disaccharide repeated m times, the monosaccharides contained in [ ]t form a disaccharide repeated t times,
m ranges from 1 to 8, t ranges from 0 to 5 and p ranges from 0 to 1, it being understood that 5xe2x89xa6m+txe2x89xa612, and their salts, in particular their pharmaceutically acceptable salts.
Advantageous compounds are the salts whose anion corresponds to formula (I.1): 
in which t represents 5, 6 or 7, and the cation is a pharmaceutically acceptable monovalent cation, as well as the corresponding acids.
The salts whose anion corresponds to formula (I.2): 
in which t represents 5, 6 or 7, and the cation is a pharmaceutically acceptable monovalent cation, as well as the corresponding acids, are also advantageous.
The salts whose anion has the formula (I.3): 
in which m represents 1, 2 or 3 and t represents 2, 3, 4 or 5, and the cation is a pharmaceutically acceptable monovalent cation, as well as the corresponding acids, are particularly advantageous.
Other preferred compounds according to the invention are those of formula (II.A): 
in which 
denotes a specific family of polysaccharides Po, linked via their anomeric carbon to Pe as defined for (I), 
is as defined for (I),
the groups OX are as defined for (I) and, for the same monosaccharide, may be identical or different,
the monosaccharide contained in [ ]mxe2x80x2 is repeated mxe2x80x2 times, the monosaccharide contained in [ ]txe2x80x2 is repeated txe2x80x2 times,and the monosaccharide contained in [ ]pxe2x80x2 is repeated pxe2x80x2 times,
mxe2x80x2 ranges from 1 to 5, txe2x80x2 ranges from 0 to 24, and pxe2x80x2 ranges from 0 to 24, it being understood that 10xe2x89xa6mxe2x80x2+txe2x80x2+pxe2x80x2xe2x89xa625, and the pharmaceutically acceptable salts thereof.
The preferred salts of the invention are those chosen from alkali metal cations and even more preferably those in which the cation is Na+ or K+.
The following polysaccharides are particularly preferred:
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)]4-O-(2,3-di-O-methyl-6-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1,4)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)]5-O-(2,3-di-O-methyl-6-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1,4)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-[O-(3-O-methyl-2,6-di-O-sulpho-xcex2-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)]6-O-(2,3-di-O-methyl-6-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(2,3-di-O-methyl-4,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3-di-O-methyl-6-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-]11-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1,4)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(2,3-di-O-methyl-4,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3-di-O-methyl-6-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-]13-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(2,3-di-O-methyl-4,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3-di-O-methyl-6-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-]15-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucbpyranosyl)-(1xe2x86x924)-[O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)]2-[O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3,6-tri-O-methyl-xcex2-D-glucopyranosyl)-(1xe2x86x924)]2-2,3-di-O-methyl-6-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-1xe2x86x924)]2-[O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3,6-tri-O-methyl-xcex2-D-glucopyranosyl)-(1xe2x86x924)]3-O-2,3-di-O-methyl-6-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3,6-tri-O-methyl-xcex2-D-glucopyranosyl)-(1xe2x86x924)]4-O-2,3-di-O-methyl-6-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3,6-tri-O-methyl-xcex2-D-glucopyranosyl)-(1xe2x86x924)]3-O-2,3-di-O-methyl-6-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex2-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-lucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)]4-O-2,3-di-O-methyl-6-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt
Methyl O-(3-O-methyl-2,4,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(3-O-methyl-2,6-di-O-sulpho-xcex2-D-glucopyranosyl)-(1xe2x86x924)-[O-(2,3,6-tri-O-methyl-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3,6-tri-O-methyl-xcex2-D-glucopyranosyl)-(1xe2x86x924)]5-O-2,3-di-O-methyl-6-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-O-(2,3-di-O-methyl-xcex1-D-glucopyranosyluronic acid)-(1xe2x86x924)-O-(2,3,6-tri-O-sulpho-xcex1-D-glucopyranosyl)-(1xe2x86x924)-(2,3-di-O-methyl-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2,3,6-tri-O-sulpho-xcex1-D-glucopyranoside, sodium salt.
The present invention relates to a process for the preparation of the compounds of formula (I), wherein, in a first step, a fully protected precursor of the desired polysaccharide (I), containing a protected precursor of the Pe region (this region being shown in Scheme 1) elongated at its non-reducing end by a protected precursor of the sulphated polysaccharide Po, is synthesized and, in a second step, the negatively-charged groups are then introduced and/or demasked. 
In a first approach, the fully protected precursor of the tetrasaccharide part EFGH of the pentasaccharide may be used. A polysaccharide Po which contains at its reducing-terminal end the missing unit D of Pe is then added in order to obtain, after coupling, the entire ABR which is thus restored.
In another approach, the fully protected precursor of the disaccharide part GH of the pentasaccharide may be used. A polysaccharide Po precursor of the TBR which contains at its reducing-terminal end the missing unit DEF of Pe is then added in order to obtain, after coupling, the entire ABR which is thus restored.
These Pe precursors are synthesized as indicated above from synthons described in the literature or forming part of the present invention.
The polysaccharide precursor part of Po is synthesized according to reactions which are well known to those skilled in the art, using the methods for synthesizing oligosaccharides (G. J. Boons, Tetrahedron, 1996, 52, 1095-1121) or an oligosaccharide when a glycosidic-linkage-donating oligosaccharide is coupled with a glycosidic-linkage-accepting oligosaccharide in order to lead to another oligosaccharide whose size is equal to the sum of the sizes of the two reactive species.
This sequence is repeated until the desired compound of formula (I) is obtained. The nature and profile of the charge of the desired final compound determine the nature of the chemical species used in the various steps of the synthesis, according to rules which are well known to those skilled in the art.
A preferred method for the preparation of the Po precursors according to the present invention is shown in Scheme 2 below: 
The term temporary is understood to refer to a substituent which is conserved for a limited number of steps, the term semi-permanent is understood to refer to a substituent which is conserved for a larger number of steps, and the term permanent is understood to refer to a substituent which is conserved to the end of the synthesis; the permanent substituents are removed during the final step. Certain permanent groups may form part of the final molecule.
In Scheme 2, (a) represents a glycosidic-linkage-donor monosaccharide in which Z is a temporary protecting group of a hydroxyl function and Y is an anomeric-carbon activator, Tn, which may be identical or different, are temporary, semi-permanent or permanent substituents of all the other hydroxyl functions.
The compound (b) which possesses an unsubstituted hydroxyl group represents a glycosidic-linkage-acceptor monosaccharide in which Tn, which may be identical or different, are temporary, semi-permanent or permanent substituents of the hydroxyl groups. T1 is a temporary, semi-permanent or permanent protecting group of the anomeric position. It is removed when it is desired to activate the anomeric carbon.
With the aim of obtaining the compounds of the invention, the glycosidic-linkage donor (a) and the glycosidic-linkage acceptor (b) react together to give the disaccharide (c).
The disaccharide (c) obtained above is converted specifically into a glycosidic-linkage-donor disaccharide (d) by removal of T1 and introduction of Y and/or into a glycosidic-linkage acceptor (e) by removal of Z.
Next, the glycosidic-linkage donor (d) and the glycosidic-linkage acceptor (e) react together to give the tetrasaccharide (f) in which t represents 1.
Repetition of this sequence of reactions gives an oligo- or a polysaccharide (f) in which t is greater than 1.
It is also possible, using the process represented in Scheme 2, to obtain a large variety of fully protected oligo- or polysaccharides such as (g) in which the oligosaccharides [ ]m and [ ]t are fully protected precursors of differently charged regions of the compounds of the invention.
In the following step of the process, the compounds such as (f) and (g) are converted into glycosidic-linkage donors and are coupled to the non-reducing terminal unit of fully protected precursors of Pe.
As has been mentioned above, the oligosaccharide of the non-reducing terminal unit of a glycosidic-linkage-donor polysaccharide (g) may constitute a part of Pe, in the case where (g) is coupled to the non-reducing terminal unit of a fully protected oligosaccharide which is the precursor of the residue of the structure of Pe.
The compounds of the invention are obtained from their fully protected polysaccharide precursors using the following sequence of reactions:
the alcohol functions which need to be converted into a sulpho group and the carboxylic acids are deprotected by removal of the Tn groups used to protect them during the development of the skeleton, then
the sulpho groups are subsequently introduced.
The compounds of the invention may, naturally, be prepared using various strategies known to those skilled in the art of oligosaccharide synthesis.
The process described above is the preferred process of the invention. However, the compounds of formula (I) may be prepared by other well-known methods of sugar chemistry described, for example, in Monosaccharides, Their chemistry and their roles in natural products, P. M. Collins and R. J. Ferrier, J. Wiley and sons, 1995 and in G. J. Boons, Tetrahedron, 1996, 52, 1095-1121.
The precursor of the part of the pentasaccharide Pe when W represents an oxygen atom and R1a is R1 is prepared according to oligosaccharide synthesis methods and particularly according to the methods described in patents EP 84,999, EP 301,618, EP 454,220 and EP 529,715 and in patent applications EP 93204769 and EP 94202470. When complete protection is carried out, it is possible, using suitable protecting groups, to obtain a free hydroxyl group on position 4 of the non-reducing terminal unit (D). The fully protected precursor of Pe is then coupled to this position using the known methods of oligosaccharide synthesis.
The pentasaccharide Pe in which W represents a carbon atom and R1a is R1, of formula: 
in which R and R1 are as defined for (I), is obtained from the synthon of formula: 
in which T1 and Tn which may be identical or different, represent a temporary, semi-permanent or permanent substituent, Z is a protecting group for a hydroxyl function which is itself obtained by a synthesis carried out by means of a radical reaction between a free-radical-generating monosaccharide and a monosaccharide containing a double bond, the C-disaccharide thus obtained then being converted into synthon (II.1) according to the standard methods described above according to C. van Boeckel and M. Petitou.
The synthon of formula (II.1), which is particularly useful in the synthesis of the compounds (II), is of formula: 
This synthon is prepared according to the reaction scheme described in Scheme 22 below.
The pentasaccharide Pe which features a substituent R1a which constitutes an L-iduronic acid unit of locked configuration, of formula: 
in which R and R1 are as defined for (I) and W represents an oxygen atom, are obtained from the synthon of formula: 
in which T1 and Tn, which may be identical or different, represent a temporary, semi-permanent or permanent substituent, Z is a protecting group for a hydroxyl function which is itself obtained by a synthesis carried out according to the methods described in the literature M. K. Gurjar et al., Tetrahedron letters, 1995, 36, 11, 1937-1940, 1933-1936 and 1994, 35, 14, 2241-2244.
The synthon of formula (III.1) which is particularly useful for synthesizing the compounds (III) is of formula: 
This synthon is prepared according to the reaction scheme described in Scheme 34 below.
The intermediates (II.1) and (III.1) are novel intermediates which are particularly useful for preparing the compounds (I) according to the invention.
The pentasaccharides Pe may thus be obtained from these disaccharide synthons (II.1) or (III.1) in the way described in the publication by C.A.A. van Boeckel and M. Petitou, Angew. Chem. Int. Ed. Engl. mentioned above.
The term semi-permanent groups used above is understood to refer to the groups which can be removed firstly after the glycosylation reactions when the carbohydrate skeleton contains the desired number of units, without removing or adversely affecting the other groups present, thereby allowing the introduction of desired functional groups into the positions they occupy.
The permanent groups are groups capable of maintaining the protection of the OH functions during introduction of the functional groups in place of the semi-permanent groups.
These groups are chosen from those which are compatible with the functional groups introduced after removal of the semi-permanent groups. They are, moreover, groups which are inert towards the reactions carried out to install these functional groups and which may be removed without these functional groups being adversely affected.
According to the invention, the permanent groups are preferably (C1-C6)alkyl groups. Examples of semi-permanent and/or temporary groups which may be mentioned are benzyl and acetyl, levulinyl, p-methoxybenzyl groups, etc.
The substituents in position 3 of the uronic units of the target compound may already be present in the starting synthons, along with the substituent R1.
The protecting groups used in the process for preparing the compounds (I) are those commonly used in sugar chemistry, for example in Protective Groups in Organic Synthesis, T W Greene, John Wiley and sons, New York, 1981.
The protecting groups are advantageously chosen, for example, from acetyl, halomethyl, benzoyl, levulinyl, benzyl, substituted benzyl, optionally substituted trityl, tetrahydropyranyl, allyl, pentenyl, tert-butyldimethylsilyl (tBDMS) or trimethylsilylethyl groups (etc.).
The activating groups are those conventionally used in sugar chemistry, for example according to G. J. Boons, Tetrahedron, 1996, 52, 1095-1121. These activating groups are chosen, for example, from imidates, thioglycosides, pentenylglycosides, xanthates, phosphites and halides.
The process described above makes it possible to obtain the compounds of the invention in the form of salts. In order to obtain the corresponding acids, the compounds of the invention in the form of salts are placed in contact with a cation-exchange resin in acidic form.
The compounds of the invention in acidic form may then be neutralized with a base in order to obtain a desired salt.
For the preparation of the salts of the compounds of formula (I), any organic or inorganic base may be used, giving, with the compounds of formula (I), pharmaceutically acceptable salts.
Sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesium hydroxide is preferably used as base. The sodium and calcium salts of the compounds of formula (I) are the preferred salts.
In step (a) of the process, the protecting groups used are those usually used by those skilled in the art of sugar chemistry, for example according to EP 84,999 or alternatively according to Protective Groups in Organic Synthesis, T W Greene, J. Wiley and sons, 1995.
The compounds (I) thus obtained may optionally be salified.
The compounds of formula (I) above also comprise those in which one or more hydrogen or carbon atoms have been replaced with their radioactive isotope, for example tritium or carbon-14. Such labelled compounds are useful in pharmacokinetic, metabolism or research studies, and in biochemical tests as ligands.
The compounds according to the invention formed the subject of biochemical and pharmacological studies which showed that they possess very advantageous properties.
The compounds of the present invention which bind selectively to AT III with an affinity equal to or greater than that of heparin possess the anticoagulant and antithrombotic properties of heparin.
The overall antithrombotic activity of the products of formula (I) was evaluated intravenously or subcutaneously in rats, in a model of venous stasis and induction by thromboplastin, according to the method described by J. Reyers et al. in Thrombosis Research, 1980, 18, 669-674 as well as in a model of arterial thrombosis consisting of a shunt implanted between the carotid artery and the jugular vein of rats, as described by Umetsu et al. Thromb. Haemost., 1978, 39, 74-83. In these two experimental models, the ED50 of the compounds of the invention is at least of the same order as or less than that of the other synthetic heparinoids already known (ED50 between 5 and 500 xcexcg/kg). The compounds of the invention thus have a specificity of action and an anticoagulant and antithrombotic activity which are particularly advantageous.
By virtue of their biochemical and pharmaceutical activity, the compounds of the present invention are very advantageous medicines. Their toxicity is entirely compatible with this use. They are also very stable and are thus particularly suitable for constituting the active principle of pharmaceutical specialty products.
Furthermore, the compounds of the invention are not neutralized by large doses of cationic platelet proteins such as platelet factor 4 (PF4) released during activation of these proteins in the process of thrombosis. The compounds of the invention are thus particularly advantageous for the treatment and prevention of thrombosis of arterial or venous origin.
They may be used in various pathologies which are consecutive to a modification of the haemostasis of the coagulation system, which appears in particular during disorders of the cardiovascular and cerebrovascular system, for instance thromboembolic disorders associated with atherosclerosis and with diabetes, such as unstable angina, cerebral attacks, restenosis after angioplasty, endarterectomy and the installation of endovascular prostheses; or thromboembolic disorders associated with rethrombosis after thrombolysis, with infarction, with dementia of ischaemic origin, with peripheral arterial diseases, with haemodialysis and with auricular fibrillations or alternatively during the use of vascular prostheses of aortocoronary bridges. These products may moreover be used for the treatment or prevention of thromboembolic pathologies of venous origin such as pulmonary embolism. They may be used for preventing or treating thrombotic complications which appear during surgical interventions or together with other pathologies such as cancer and bacterial or viral infections. When they are used during the installation of prostheses, the compounds of the present invention may coat the prostheses and thus make them haemocompatible. In particular, they may be bound to intravascular prostheses (stents). In this case, they may optionally be chemically modified by introduction of a suitable arm onto the reducing or non-reducing end, as described in EP 649,854.
The compounds of the present invention may also be used as adjuvants during endarterectomy performed with small porous balloons.
The compounds of the invention are very stable and are thus particularly suitable for constituting the active principle of medicines.
According to another of its aspects, the subject of the present invention is thus a pharmaceutical composition containing, as active principle, a synthetic polysaccharide as defined above.
The invention preferably relates to pharmaceutical compositions containing, as active principle, a compound of formula (I), (I.1), (I.2) or (I.3) or one of its pharmaceutically acceptable salts, optionally in combination with one or more inert and suitable excipients.
In each dosage unit, the active principle is present in amounts adapted to the daily doses envisaged. In general, each dosage unit is conveniently adjusted according to the dosage and type of administration planned, for example tablets, gelatin capsules and the like, sachets, ampules, syrups and the like, drops and transdermal or transmucous patches, such that one dosage unit contains from 0.1 to 100 mg of active principle, preferably 0.5 to 50 mg.
The compounds according to the invention may also be used in combination with another active principle which is useful for the desired therapy such as, for example, antithrombotic agents, anticoagulants or platelet-antiaggregating agents, for example such as dipyridamole, aspirin, ticlopidine, clopidogrel or antagonists of the glycoprotein IIb/IIIa complex.
The pharmaceutical compositions are formulated for administration into mammals, including man, for the treatment of the abovementioned diseases.
The pharmaceutical compositions thus obtained are advantageously in various forms such as, for example, injectable or drinkable solutions, tablets, coated tablets or gelatin capsules. The injectable solutions are the preferred pharmaceutical forms. The pharmaceutical compositions of the present invention are useful in particular for the preventive or curative treatment of disorders of the vascular wall, such as atherosclerosis, the hypercoagulability states observed, for example, after surgical operations, tumour development or deregulation of coagulation, which are induced by bacterial, viral or enzymatic activators. The dosage may vary widely as a function of the age, weight and state of health of the patient, the nature and severity of the complaint and the route of administration. This dosage comprises the administration of one or more doses of from 0.1 mg to 100 mg per day approximately, preferably from 0.5 to 50 mg per day approximately, intramuscularly or subcutaneously, in continuous administrations or administrations at regular intervals.
The subject of the present invention is thus also pharmaceutical compositions which contain, as active principle, one of the above compounds optionally combined with another active principle. These compositions are prepared so as to be able to be administered via the digestive or parenteral route.
In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, transmucous, local or rectal administration, the active ingredient may be administered in unit forms of administration, mixed with standard pharmaceutical vehicles, to animals and to man. The appropriate unit forms of administration comprise oral forms such as tablets, gelatin capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, subcutaneous, intramuscular, intravenous, intranasal or intraoccular administration forms and rectal administration forms.
When a solid composition in tablet form is prepared, the main active ingredient is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets may be coated with sucrose or other suitable materials or alternatively they may be treated such that they have a sustained or delayed activity and so that they release a predetermined amount of active principle continuously.
A preparation in gelatin capsules is obtained by mixing the active ingredient with a diluent and pouring the mixture obtained into soft or hard gelatin capsules.
The water-dispersible powders or granules may contain the active ingredient mixed with dispersing agents or wetting agents, or suspending agents, for instance polyvinylpyrrolidone, as well as with sweeteners or flavour enhancers.
For rectal administration, use is made of suppositories which are prepared with binders that melt at rectal temperature, for example cocoa butter or polyethylene glycols.
For parenteral, intranasal or intraoccular administration, sterile, injectable solutions, isotonic saline solutions or aqueous suspensions which contain pharmacologically compatible dispersing agents and/or wetting agents, for example propylene glycol or butylene glycol, are used.
For transmucous administration, the active principle may be formulated in the presence of a promoter such as a bile salt or in the presence of a hydrophilic polymer such as, for example, hydroxypropylcellulose, hydroxypropylmethylcelluose, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, pectins, starches, gelatin, casein, acrylic acids, acrylic esters and their copolymers, vinyl polymers or copolymers, vinyl alcohols, alkoxypolymers, polyethylene oxide polymers and polyethers, or a mixture thereof.
The active principle may also be formulated in the form of microcapsules, optionally with one or more vehicles or additives.
The active principle may also be in the form of a complex with a cyclodextrin, for example xcex1-, xcex2- or xcex3-cyclodextrin, 2-hydroxypropyl-xcex2-cyclodextrin or methyl-xcex2-cyclodextrin.
The active principle may also be released by a small balloon containing it or by an endovascular expander introduced into blood vessels. The pharmacological efficacy of the active principle is thus not adversely affected.
Subcutaneous administration is the preferred route.
The following methods, preparations and schemes illustrate the synthesis of the various intermediates which are useful for obtaining the polysaccharides according to the invention.
The examples below also illustrate the invention without, however, limiting it.
The following abbreviations are used:
TBDMS: tert-butyldimethylsilyl; Lev: levulinyl; Bn: benzyl; Bz: benzoyl; TLC: thin-layer chromatography; Olm: trichloroacetimidyl; LSIMS: Liquid Secondary Ion Mass Spectrometry; ESIMS: Electron Spray Ionization Mass. Spectrometry; TMS: trimethylsilyl; TSP: sodium trimethylsilyltetradeuteriopropionate; Tf: triflate; MS: molecular sieves; All: allyl; PMB: p-methoxybenzyl; SE: trimethylsilylethyl.
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